Successful and efficacious mucosal protein delivery requires protection, sustained release, and conformational maintenance of encapsulated proteins. Poly (lactide coglycolite) (PLG), poly (lactide), and poly (anhydride), have been used to generate a matrix for microparticulates1. Unfortunately, the use of these polymers can result in an acidic microenvironment during formulation and biodegradation of these microspheres, and co-valent reactions between the polymer or its degradation products with encapsulated peptides/proteins also a potential threat to protein integrity. Keeping these points in perspective, the present investigation describes the preparation of microspheres (1 to 10 microns) that were prepared by a novel polymer dispersion technique and coated with chitosan and poly caprolactone (PCL) to prevent interaction with the alginate matrix, protect against enteric degradation and extend the release of recombinant proteins. Our preliminary study shows that Alginate microspheres (PACEA) release their entrapped proteins over 90 days and also demonstrated the use of alginate microspheres for oral vaccine delivery of tetanus toxoid (TT). After a single oral dose of alginate microsphere encapsulated TT, formulated to release contents at 7, 30, and 90 days, mice developed protective serum Ab titers against TT. These studies provide the rationale for our working hypothesis that entrapped proteins can be protected and maintain the native protein conformation which would affect their therapeutic efficacy. Protective mucosal and systemic adaptive immunity against pneumococcal carriage and pneumonia can be induced by oral delivery of alginate microsphere- encapsulated PsaA. We have emphasized in-vitro as well as in vivo approaches using mouse models of pneumococcal -carriage pneumonia to test this hypothesis. Aim One will assess the stability of alginate microsphere-encapsulated PsaA vaccines by the various process methods. Aim Two will characterize the particle uptake by mucosal and systemic adaptive organs and their stability to alginate microsphere encapsulated GFP. Aim Three will ascertain the ability of alginate microsphere encapsulated PsaA vaccines to induce protective immunity against S. pneumoniae challenge, using EF3030 in mice. This study will provide important and new information regarding the cellular and molecular mechanisms of oral delivery of proteins and vaccines by alginate microspheres against pneumococci. Orally effective vaccines have been previously developed against various infective agents, e.g., polio. More recently, vaccines administered by the oral and intranasal routes have shown great promise against both intestinal and respiratory tract infections. This study addresses an important health problem since pneumococci in nasopharyngeal carriage are thought to be the main human reservoir for this potential-lethal organism. Understanding the cellular and molecular mechanisms of mucosal pneumococcal immunity is important for understanding and devising ways to protect against carriage, which is considered to be essential for herd immunity to Streptococcus pneumoniae. This Gram-positive bacterium is a major cause of acute otitis media, pneumonia, bacteremia, and meningitis. Pneumococcal pneumonia is among the top ten causes of death in aged populations hence, vaccination against pneumococcal infections is greatly needed. Therefore, selection of appropriate pneumococcal antigens and mucosal adjuvants will greatly improve the efficacy of future vaccines. Moreover, single (or multiple) oral vaccination would promote both mucosal and systemic immunity and no doubt improve compliance. Further, single-dose vaccines would result in enhanced immunity due to increases in compliance and the ease of administration. This application presents a novel method of polycaprolactone (PCL)- and chitosan-coated epichlorohydrin-crosslinked alginate (PACE-A) microspheres for vaccine delivery. [unreadable] [unreadable] [unreadable]